The seven terminal steps involved in synthesis of bacteriophage T4 tRNAPro and tRNASer have been defined by mutant methodology and confirmed by a reconstruction of the reactions in vitro from purified precursor RNA intermediates and enzymes. The next phase of this work will be directed at obtaining a more comprehensive understanding of the molecular features underlying the specificity of precursor RNA-enzyme interactions. This effort is likely to be assisted by the availability of mutants altered in specific interactions. An additional effort will be made to extend the synthetic pathway back toward the DNA molecule, to include a definition of the steps responsible for nucleotide modifications and cleavage reactions that lead to the precursor RNA molecules thus far identified. Another goal of this project is concerned with investigating the evolution of tRNA genes. Using a system amenable to genetic manipulation and biochemical analysis, we will determine if a tRNA gene can evolve from complementary segments of two parental tRNA genes. Also we will evaluate the proposition that a process of this type was responsible for creation of ribosomal RNA genes. Mutant strains with high reversion frequencies are not uncommon. Geneticists are interested in such mutant strains because they may represent important mutational events. We intend to investigate a set of unstable mutants of a bacteriophage t4 tRNA gene with the aim of characterizing the mutants for their nucleotide alterations, mode of reversion, and enzymology of the reversion process.